Disorders of protein metabolism in skeletal muscle are central to the pathogenesis of diabetes and other disease states that affect nutrient homeostasis. Development of rational approaches to treatment of these disorders requires knowledge of the basic events involved in the regulation of protein metabolism. Therefore, the overall goal of the project is to provide a better understanding of the mechanisms by which insulin regulates specific events in the protein synthetic pathway in skeletal muscle. Studies proposed for the next project period are designed to elucidate the mechanism of insulin action on the first event in the translational phase of protein synthesis, i.e., formation of a 43S initiation complex composed of eukaryotic initiation factor eIF-2, GTP, initiator methionyl-tRNA, and a 40S ribosomal subunit. Additionally, they are designed to provide new insights into a role for insulin in the regulation of pretranslational events in skeletal muscle. The specific aims of the studies are: (1) to further characterize effect of insulin on eIF-2, particularly with respect to covalent modifications, intracellular distribution, and interaction with the guanine nucleotide exchange factor (GEF); (2) to establish and characterize muscle cell cultures in which insulin regulates peptide-chain initiation and from which can be prepared an active cell-free, protein-synthesizing system; (3) to investigate effects of insulin on activity and content of eIF-2 and GEF, and relate their content to number of ribosomes and rate of protein synthesis; (4) to clone cdna sequences coding for the alpha- and beta-subunits of eIF-2, to derive the amino acid structure of the subunits from sequence analysis of the cDNA inserts, and to employ the cDNA clones to quantitate the amount of mRNA for the alpha- and beta-subunits; and (5) to investigate effects of insulin on total and selected mRNA species, and to clone cDNA sequences coding for proteins that show large and rapid responses to insulin. The studies are to utilize a number of methodologies including muscle perfusion, cell culture, cell-free protein- synthesizing systems, protein purification, immunochemical identification and quantitation of proteins, one- and two- dimensional gel electrophoresis, and quantitation of mRNA using specific cDNAs. Overall, the studies described in this proposal should help identify mechanisms by which insulin regulates protein synthesis in skeletal muscle. They should also provide new insights into biochemical events involved in peptide-chain initiation.